The emerging bioterrorism threat has galvanized the need for rapidly effective treatments against botulism. This acute neuroparalytic condition is produced by botulinum neurotoxin (BoNT), which ranks among the most deadly toxins known. The BoNT light chain (BoNT-LC) is a highly specific endopeptidase that inhibits neurotransmitter release from presynaptic membranes inside human neuronal cells. To meet the challenge posed by the rapid intracellular entry of BoNT, experiments are proposed to develop recombinant BoNT inhibitors that can be swiftly delivered to the neuronal-cell cytosol. In Aim 1, we will identify single-chain fragment variable antibodies that trap the LC of BoNT (serotype B) and prevent its functional maturation. Variants of this prototype inhibitor will be engineered that inactivate BoNT/B by blocking access to the catalytic site of BoNT/B-LC (Aims 2 & 3). We will enable the inhibitors to the cross plasma membrane using a proprietary intracellular delivery system, thus generating "intracellular toxin traps" that bind to and inactivate BoNT within the intracellular compartment where the peptidase normally operates. These highly integrated studies will generate a new type of intracellular therapeutic and fill an existing gap in currently available measures to counteract the lethal effects of BoNT. [unreadable] [unreadable]